Evaluation of neuroretina following i.v. or intra‐CSF AAV9 gene replacement in mice with MPS IIIA, a childhood dementia

Abstract Background Sanfilippo syndrome (mucopolysaccharidosis type IIIA; MPS IIIA) is a childhood dementia caused by inherited mutations in the sulfamidase gene. At present, there is no treatment and children with classical disease generally die in their late teens. Intravenous or intra‐cerebrospinal fluid (CSF) injection of AAV9‐gene replacement is being examined in human clinical trials; evaluation of the impact on brain disease is an intense focus; however, MPS IIIA patients also experience profound, progressive photoreceptor loss, leading to night blindness. Aim To compare the relative efficacy of the two therapeutic approaches on retinal degeneration in MPS IIIA mice. Methods Neonatal mice received i.v. or intra‐CSF AAV9‐sulfamidase or vehicle and after 20 weeks, biochemical and histological evaluation of neuroretina integrity was carried out. Results Both treatments improved central retinal thickness; however, in peripheral retina, outer nuclear layer thickness and photoreceptor cell length were only significantly improved by i.v. gene replacement. Further, normalization of endo‐lysosomal compartment size and microglial morphology was only observed following intravenous gene delivery. Conclusions Confirmatory studies are needed in adult mice; however, these data indicate that i.v. AAV9‐sulfamidase infusion leads to superior outcomes in neuroretina, and cerebrospinal fluid‐delivered AAV9 may need to be supplemented with another therapeutic approach for optimal patient quality of life.


| INTRODUC TI ON
Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) is a childhood dementia-causing disorder that is presently untreatable.It arises due to autosomal recessive mutations in the sulfamidase (SGSH) gene encoding a lysosomal sulfatase involved in the stepwise degradation of heparan sulfate (HS), leading to its accumulation in lysosomes, with cell dysfunction and death ensuing. 1 Children with the classical form of the disease exhibit early and progressive cognitive impairment, sleep difficulties, autistic traits, motor dysfunction, and vision loss.The median age of death in children with rapidly progressing MPS IIIA is 18 years. 2eatment is mostly supportive; however, a variety of potential therapeutics either have been or are currently being evaluated in human clinical trials.The most active areas of research are recombinant human enzyme e.g., Refs.[3,4] or gene replacement, while substrate inhibition and targeting of the innate immune response are also being examined (www.clini caltr ials.gov NCT01299727; 02053064; 03300453; 03423186; 03612869; 04018755; 04088734).Viral vectors used for gene therapy in patients include AAVrh10human sulfamidase delivered via multisite intra-cerebral infusion (NCT01474343; NCT02053064; NCT03612869), 5,6 scAAV9-U1asulfamidase delivered via intravenous (i.v.) injection (NCT02716246; NCT04088734; NCT04360265) 7 and AAV9-CAG-coh-sulfamidase delivered via intra-ventricular cerebrospinal fluid (CSF) injection (EudraCT: 2015-000359-26). 8,9The human clinical trials are ongoing, and no data has been published in the peer-reviewed literature to date, to our knowledge.Further, direct comparison of the two AAV9-sulfamidase delivery methods has not been undertaken to the best of our understanding; however, preclinical data indicate that both routes of injection lead to significant reductions in brain pathology and improved cognitive function in treated mice. 7,8ss is known about the impact of the therapies on the retina, however.5][16][17] Significant early endo-lysosomal expansion occurs in MPS IIIA mouse retina leading to changes in microglial morphology by 3-6 weeks of age. 14 Improvements in retinal pathology were reported after i.v.

AAV9
-sulfamidase delivered to one-month old MPS IIIA mice by Fu et al. 7 Whether intra-CSF AAV9-sulfamidase prevents or slows retinal disease lesion formation has not been reported to date.To enable a head-to-head comparison of the two clinically relevant routes of delivery of AAV9-sulfamidase, we performed intra-CSF infusions of vector into the lateral ventricles of neonatal MPS IIIA mice and the impact on retinal disease at 20 weeks of age was compared to that observed following i.v.delivery of the same vector at the same disease stage.Our rationale was that should the two putative therapeutic approaches prove equivalent in preventing/slowing cognitive decline in human clinical trials, their respective efficacy in nervous system tissues other than brain may identify one of the two routes of administration as a strategy of choice, given that the goal of therapy is to maximize both the quantity and quality of life of patients with MPS IIIA.

| RE SULTS
Groups of conscious male/female pups received treatment with the AAV9-CMV-human sulfamidase vector via injections made into the superficial temporal vein (referred to hereafter as i.v.) and tail vein.
Infusions were also made into ventricular CSF (referred to hereafter as intra-CSF) in additional cohorts of cryoanaesthetised pups.The study protocol is shown in Figure 1.Significant loss of MPS IIIA pups was observed following intra-CSF infusions, regardless of infusate type (PBS as vehicle or vector).Of fifteen mice per group, only 5-6 per group remained at weaning.Death of wildtype vehicle-treated mice also occurred, but to a lesser degree (13 of 15 mice survived to weaning).Litter loss has previously been reported in MPS IIIA mouse colonies 18 and is increased when mice are disturbed. 19In the cohorts of mice/group receiving i.v.infusions, only one litter of six MPS IIIA mice that had been injected with PBS was cannibalized.As one MPS IIIA mouse eye from the intra-CSF AAV9-sulfamidase treatment group was damaged upon retrieval and not evaluated further, group sizes for analysis at 20 weeks of age varied from n = 4-13.Data from individual mice are presented throughout.

| i.v. delivered AAV9-sulfamidase normalizes endo-lysosomal compartment size
As shown in Figure 2A,B, MPS IIIA mice exhibit greatly reduced sulfamidase activity in retina compared to wildtype and increases in enzyme activity were only seen in mice treated via i.v.AAV9sulfamidase.Intravenous infusion of vector normalized HS levels (Figure 2C), whereas significant amounts of HS remained in intra-CSF AAV9-sulfamidase treated MPS IIIA mouse retina (Figure 2D).
Compatible with the HS outcomes, staining of the endo-lysosomal compartment with antibodies to lysosomal integral membrane protein 2 (LIMP2; Figure 3) revealed that while intra-CSF AAV9sulfamidase significantly reduced the size of the compartment, only i.v.AAV9-sulfamidase treatment was able to normalize it.As expected based on previously published studies, i.v.AAV9-sulfamidase treatment increased brain SGSH activity, but to a lower level than that seen following intra-CSF infusion of AAV9-sulfamidase (18.6% of unaffected vs. 97.7% of unaffected SGSH activity, respectively; Figure S1).These observations are compatible with the relative amount of hSGSH mRNA transcripts that were detectable in brain following treatment via each route, with ~22× more transcript recorded in the brain of mice treated via intra-CSF infusion (Figure S1).
Treatment with AAV9-sulfamidase via both routes completely normalized HS levels in brain. 20,21

| Impact of the two treatments on the integrity of individual retinal cell layers
As shown in Figure 4, total retinal thickness nearer the optic nerve (central) is decreased in MPS IIIA mice, mostly due to outer nuclear layer (ONL) thinning following photoreceptor segment (PS) loss.Both treatments prevented loss of photoreceptors in this region of retina, and the thickness of the retinal layers was not different from that seen in control/unaffected mice.In contrast, when measurements were taken further away from the optic nerve (peripheral; Figure 5), photoreceptor loss was unable to be prevented by intra-CSF AAV9sulfamidase but was completely prevented by i.v.AAV9-sulfamidase.

| i.v. but not intra-CSF AAV9-sulfamidase leads to retention of rhodopsin-positive photoreceptors across retina
Rhodopsin staining of rod photoreceptors revealed that outer segment (vs.inner segment) loss is pronounced in the retina of MPS IIIA mice (Figure 6) and was prevented in central retina by gene replacement delivered via both routes.Consistent with the above results, only i.v.AAV9-sulfamidase was able to prevent the loss of outer segments in peripheral retina.

| There are fewer isolectin-B4-reactive microglia in retinae of mice treated with i.v. AAV9 versus intra-CSF AAV9
Large numbers of isolectin B4-positive microglia with an ameboid morphology are noted in 20-week-old MPS IIIA mouse retina (Figure 7).i.v.AAV9-sulfamidase prevented development of these morphological changes in all retinal layers.In contrast, intra-CSF AAV9-sulfamidase was only able to partially improve the microglial phenotype in the photoreceptor layer and similar numbers of ameboid isolectin-B4-reactive microglia were seen in the GCL, IPL, and OPL of intra-CSF vehicle-and intra-CSF AAV9-sulfamidase-treated mice.

| DISCUSS ION
This study sought to compare the ability of two clinically relevant viral vector delivery approaches in preventing retinal disease in MPS IIIA mice.We have previously demonstrated that the AAV9based gene therapy strategies used here normalize HS in MPS IIIA mouse brain at 20 weeks (Shoubridge, PhD Thesis). 21Previous studies have also demonstrated that retinal degeneration in murine MPS IIIA begins early-at around six-weeks of age, with significant and progressive loss of photoreceptor outer segments. 14Elevated levels of HS and the appearance of ameboid-shaped isolectin-B4positive microglia are detectable by three-weeks of age 14 ; thus, in the present proof-of-principle study, we sought to prevent retinal F I G U R E 1 Overview of the study.Mice were injected on days 0-1 of life (n = 15-16/group).Injections were made intravenously (i.v.), via the superficial temporal vein or intra-CSF, via the lateral ventricles.Mice treated with i.v.AAV9 received a second injection on day 5 of life.At 20 weeks of age, mice were euthanased and the eyes were removed and processed for quantification of sulfamidase activity, heparan sulfate accumulation, and for immunohistochemical or histochemical evaluation of the impact of treatment on neuroretina (n = 4-13/group).Created with BioRender.comdegeneration by providing human sulfamidase gene replacement to mice at birth.We also note that MPS IIIA patient clinical trials have recently begun restricting entry to infants under 24 months (e.g., NCT04201405, NCT02716246), thus treatment of very young subjects is appropriate.
We used a single vector design-single-strand AAV9-CMVhuman sulfamidase but treated some mice via intravenous infusion and other mice via intra-ventricular CSF injection.These two delivery routes are currently being examined as strategies for providing AAV9-based gene therapy to children with MPS IIIA (NCT02716246 and EudraCT 2015-000359-26, respectively).
Given that most of the vector genomes end up in the liver following i.v.infusion (e.g., Ref. [7]) and only 5%-10% of vector genomes are detected in brain, we gave larger amounts of vector to mice receiving i.v.infusion.Our dosing strategy is also compatible with the range of doses provided clinically.Providing equivalent vector genomes i.v. and intra-CSF would undoubtably lead to either vector-mediated side effects, e.g., reactive gliosis in brain if the dose given intra-CSF was larger to match that provided i.v., or insufficient vector reaching central nervous system if the dose given i.v. was lower to match that given intra-CSF.As expected, despite the disparity in vector dose, much lower amounts of hSGSH transcript and SGSH activity were detected in brain following i.v.versus intra-CSF infusion of AAV9-SGSH.In support of this, central retina versus medial or peripheral retina, was preferentially treated when mice with CLN6 deficiency which causes late infantile Batten disease, received intra-CSF AAV9-based gene replacement. 22Limited gene/protein expression in retina following injection of AAV vectors into the CSF has also been observed previously in additional models of Batten disease-sheep with CLN5 deficiency 23 and dogs with TPP1 deficiency. 24,25 higher vector dose does not explain this outcome based on the brain data, we hypothesize that the localized treatment of retina mediated by intra-CSF injection of vector is due to the way the AAV9 particles or potentially the brain-expressed human sulfamidase reaches the retina-via retrograde transport in ganglion cell axons in the optic nerve.AAV9 has been shown to undergo bidirectional transport in neurons, with retrograde transport occurring in a dynein-dependent way. 26,27Interestingly, intravitreal injection of AAV2β-glucuronidase enabled this lysosomal enzyme to be transported anterogradely along the optic nerve into select murine brain regions. 28Vector genomes were not detectable in the brain of AAV2β-glucuronidase-treated mice.Thus, it is possible that the human sulfamidase produced in brain following intra-CSF injection of AAV9-sulfamidase has been transported retrogradely in retinal ganglion cell axons to the cell bodies in the retina, along with vector particles-but the treatment effect is limited to regions proximal to optic nerve transection, annotated as "central retina" in our study.
Systemically delivered AAV9, in contrast, has been shown to cross the neonatal mouse blood-retinal barrier via transcytosis, 29 and has also been documented to access adult mouse retina following i.v.infusion. 30While studies in older animals are required for confirmation, if the outcomes in humans are comparable to those made here and elsewhere in mice, sheep, and dogs, [22][23][24][25] i.v.delivery of AAV9 may be the route of choice as it appears to enable most if not all neural and non-neural tissues to be treated (Fu et al. 7 ; this study).
In contrast, Haurigot et al. 8 and the abovementioned Batten disease model studies have shown that intra-CSF infusion of AAV9-based gene replacement leads to transduction of brain, spinal cord, and peripheral nervous system, e.g., peripheral ganglia, together with other organs; however, there is inferior access to, and thus treatment of, retina.Thus, another strategy is likely to be required for treating retinal abnormalities in patients treated with intra-CSF-directed gene replacement, for instance direct intraocular gene therapy.This resulted in significant improvements in retinal structure and function as demonstrated in MPS VII mice 31 and CLN5-deficient sheep. 32nally, it is important to note that there are differences be- of retinal transduction and the speed and amount of gene expression.First, the promotor in the vector being administered in the clinical trial of i.v.gene therapy is a U1a promoter, rather than the CMV promoter used here.This constitutive small nuclear RNA promoter (U1a) appears to enable persistent high-level gene expression in a pan-cellular fashion. 33Further, the small size of this   promotor is compatible with the smaller packaging capacity of selfcomplementary AAV vectors such as that used in the i.v.gene therapy trial in MPS IIIA patients.Use of an scAAV9 vector may also hasten gene expression (e.g., Ref. [34]) compared to single-strand AAV9 vectors like the one used here.
The promotor being used in the clinical trial of intra

| Vector
The single strand AAV9-CMV-human sulfamidase vector was synthesized by Vector Labs (Malvern, PA USA) and provided at a concentration of 9.2 × 10 13 genome copies per mL.

| Mice
The mice used in this study were sourced from breeding colonies

| Mouse injections
i.v.injections were made into awake pups via the superficial temporal vein on day 1 and the tail vein on day 5 of life (Figure 1).On each occasion, 5 × 10 11 vector genomes were delivered in 10 μL (dosing strategy as per Byrne et al. 29 ).Intraventricular CSF injections were given on day 0 of life to cryoanaesthetised pups.
Briefly, this involved cryoanaesthesia of mice followed by transil- ).The twenty-fold difference in vector genomes delivered accounts for the fact that only ~5%-10% of vector genomes reach the central nervous system (i.e., brain) compared with liver following i.v.injection. 7

F I G U R E 2
Effect of neonatal i.v.(A, C) or intra-CSF (B, D) delivery of AAV9-CMV-sulfamidase on sulfamidase activity (A, B) and HS accumulation (C, D) in 20-week-old MPS IIIA mouse retina.IS = internal standard.**p < 0.01, ***p < 0.001, ****p < 0.0001.Only statistically significantly different comparisons between groups are shown.While both injection routes facilitate sulfamidase delivery to central retina, for the first time we demonstrate that i.v. but not intra-CSF infusions of AAV9-CMV-sulfamidase enable treatment of peripheral retina in MPS IIIA, a site distant from the optic nerve.

4 F I G U R E 5
the vector used here (AAV9-CMV-human sulfamidase) and the vectors being administered in trials of i.v. and intra-CSF AAV9sulfamidase in children with MPS IIIA, that may affect the degree F I G U R E 3 Effect of neonatal i.v. or intra-CSF delivery of AAV9-CMV-sulfamidase on endo-lysosomal compartment size in 20-weekold MPS IIIA mouse retina.Lysosomal integral membrane protein 2 (LIMP2) staining from the GCL to the external limiting membrane was assessed in both central and peripheral retina (A-D).Representative photos demonstrating the staining in the peripheral retina are provided for i.v.(E) and intra-CSF delivery (F).Ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), external limiting membrane (ELM, dashed), photoreceptor segments (PS), retinal pigmented epithelium (RPE).Scale bar = 25 μm.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Only statistically significantly different comparisons between groups are shown.Effect of neonatal i.v. or intra-CSF delivery of AAV9-CMV-sulfamidase (or vehicle) on central retinal thickness in 20-weekold mice.The thickness of total retina from the GCL to but not including the RPE was measured in H&E-stained sections (A, B), as was the thickness of individual retinal layers (C-L).Representative photos show total retina in i.v. or intra-CSF treated mice (M, N respectively) or ONL only (O, P respectively).Ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor segments (PS), inner segments (IS), and retinal pigmented epithelium (RPE).Scale bar in M, N is 50 μm.Scale bar in O, P is 10 μm.**p < 0.01, ***p < 0.001, ****p < 0.0001.Only statistically significantly different comparisons between groups are shown.Effect of neonatal i.v. or intra-CSF delivery of AAV9-CMV-sulfamidase (or vehicle) on peripheral retinal thickness in 20-weekold mice.The thickness of the retina from the GCL to but not including the RPE was quantified in H&E-stained sections (A, B).The thickness of individual retinal layers was also evaluated (C-L).Photos of the total retina (M, N) or ONL (O, P) in representative mice from each treatment group are shown.Ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor segments (PS), inner segments (IS), and retinal pigmented epithelium (RPE).Scale bar in M, N is 20 μm.Scale bar in O, P is 10 μm.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Only statistically significantly different comparisons between groups are shown.
lumination of the head using a MicroLight 150 Cold Light Fiber Optic Illuminator (Fibreoptic Lightguides, VIC, Australia) and infusion of vector through a 27G dental needle held in the arm of a stereotaxic (David Kopf Instruments, CA, USA).The needle was attached to polyethylene tubing (Becton Dickinson, USA), connected to a Hamilton syringe which was placed on a slow infusion pump (SP200iz Syringe Infusion Pump (World Precision Instruments, Sarasota, FL, USA)), permitting controlled infusion of vector (1 μL/min).Injections were made at the mid-point between lambda and bregma, 1 mm lateral to the sagittal suture and 2 mm deep.Mice recovered under a warm lamp.A total of 5 × 10 10 vector genomes was delivered in 2 μL (1 μL per hemisphere; dose as per Haurigot et al.8 Loss (or reduction in length) of rod photoreceptor outer segments is noted in central MPS IIIA mouse retina by six weeks of age, but inner segment loss was not apparent until 20 weeks of age.Impaired electroretinogram recordings are evident from ~12 weeks of age in MPS IIIB mice, 15 with progressive deterioration noted thereafter.

S AND ME THODS 4.1 | Approvals
In summary, our findings indicate that intra-CSF viral vectorbased treatment of MPS IIIA patients will potentially need to be supplemented with an intraocular therapeutic to modify the profound and progressive retinal dysfunction seen in this disorder.Patients receiving i.v.AAV9-sulfamidase may not need supplementary treatment of retinal disease if these findings in neonatal mice are translatable to young children.Evaluation and reporting of whether retinal dysfunction is halted or prevented in children treated with i.v.AAV9sulfamidase (or not) is urgently needed.While the improvement or maintenance of cognitive function is of supreme importance when assessing the efficacy of a therapeutic in MPS IIIA patients, for maximum quality of life, the prevention of vision loss also requires increased focus.4| MATERIAL Sgsh mps3a ) and congenic MPS IIIA mice crossed with a reporter strain (B6.Cg-Tg(Thy1-YFP)HJrs/J; JAX Stock #003782) were used.All mice were group-housed in a temperature/humidity-controlled facility with a 14-h light: 10-h dark cycle.Food and water were available ad libitum.The mice received toilet rolls and plastic cups plus nesting material for enrichment purposes.All breeding, housing, and experimental procedures complied with the Association for Research in Vision and Ophthalmology (ARVO) statement and the Australian code for the care and use of animals for scientific purposes (8th edition; 2013).
maintained at the Women's and Children's Hospital Network, North Adelaide, South Australia, Australia.Congenic MPS IIIA mice (B6.Cg-